ToxSci Advance Access originally published online on November 8, 2006
Toxicological Sciences 2007 95(2):452-461; doi:10.1093/toxsci/kfl162
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Perfluorooctanoic Acid and Perfluorononanoic Acid in Fetal and Neonatal Mice Following In Utero Exposure to 8-2 Fluorotelomer Alcohol
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* Interdisciplinary Toxicology Program, College of Public Health, University of Georgia, Athens, Georgia 30602
Center for Food Safety, College of Agricultural and Environmental Sciences, University of Georgia, Athens, Georgia 30602
1 To whom correspondence should be addressed at National Exposure Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, 960 College Station Road, Athens, GA 30605. Fax: (706) 355-8202. E-mail: Henderson.matt{at}epa.gov.
Received August 23, 2006; accepted November 1, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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8-2 Fluorotelomer alcohol (FTOH) and its metabolites, perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA), are developmental toxicants but metabolism and distribution during pregnancy are not known. To examine this, timed-pregnant mice received a single gavage dose (30 mg 8-2 FTOH/kg body weight) on gestational day (GD) 8. Maternal and neonatal serum and liver as well as fetal and neonatal homogenate extracts were analyzed using gas chromatography coupled with mass spectrometry. During gestation (GD9 to GD18), maternal serum and liver concentrations of PFOA decreased from 789 ± 41 to 668 ± 23 ng/ml and from 673 ± 23 to 587 ± 55 ng/g, respectively. PFOA was transferred to the developing fetuses as early as 24-h posttreatment with concentrations increasing from 45 ± 9 ng/g (GD10) to 140 ± 32 ng/g (GD18), while PFNA was quantifiable only at GD18 (31 ± 4 ng/g). Post-partum, maternal serum PFOA concentrations decreased from 451 ± 21 ng/ml postnatal day (PND) 1 to 52 ± 19 ng/ml (PND15) and PFNA concentrations, although fivefold less, exhibited a similar trend. Immediately after birth, pups were cross-fostered with dams that had been treated during gestation with 8-2 FTOH (T) or vehicle (C) resulting in four treatment groups in which the first letter represents in utero (fetal) exposure and the second represents lactational (neonatal) exposure: C/C, T/C, C/T, T/T. On PND1, neonatal whole-body homogenate concentrations of PFOA from T/T and T/C groups averaged 200 ± 26 ng/g, decreased to 149 ± 19 ng/g at PND3 and this decreasing trend was seen in both neonatal liver and serum from PND3 to PND15. Based on detectible amounts of PFOA in neonatal serum in the C/T group on PND3 (57 ± 11 ng/ml) and on PND15 (58 ± 3 ng/ml), we suggest that the neonates were exposed through lactation. In conclusion, exposure of neonates to PFOA and PFNA occurs both pre- and postnatally following maternal 8-2 FTOH exposure on GD8.
Key Words: fluorotelomer alcohols (FTOH); PFOA; PFNA; fetal and neonatal distribution.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fluorotelomer alcohols (FTOHs) are used in the manufacture of fluorinated surfactants and polymers and in the surface-active modification of consumer products. They are linear chain polyfluorinated alcohols in which the –OH moiety links the polyfluorinated alkyl tail to various polymers via ester, amide, urethane, and ether linkages. Furthermore, the FTOHs are used as intermediates in the synthesis of dyes, paints, adhesives, polymers, and waxes (Dinglasan-Panlilio and Mabury, 2006
). The FTOHs have been shown to be present in the North American troposphere at concentrations up to 165 pg/m3 (Stock et al., 2004) and it is estimated that the global production from 2000 to 2002 exceeded 5000 tons/year (Prevedouros et al., 2006).
Following release, 8-2 FTOH and structurally analogous compounds are likely transported via atmospheric processes and subsequently transformed into perfluorooctanoic acid (PFOA) via both metabolism and biodegradation processes (Fasano et al., 2006; Kudo et al., 2005; Martin et al., 2005). Perfluorinated carboxylic acids (PFCAs) are formed during atmospheric oxidation of FTOHs (Ellis et al., 2004; Wallington et al., 2006). PFOA was formed by aerobic degradation of 8-2 FTOH in a mixed microbial system (Dinglasan et al., 2004; Wang et al., 2005), and via mammalian metabolism of these precursor compounds (Fasano et al., 2006; Kudo et al., 2005; Martin et al., 2005). However, PFCAs including PFOA and perfluorononanoic acid (PFNA) are resistant to metabolism and environmental biodegradation reactions including oxidation, hydrolysis, and reductive halogenation. Both PFOA and PFNA are of particular concern because they are ubiquitous in both human and environmental matrices (Martin et al., 2002; Olsen et al., 2003; Stock et al., 2004; Taniyasu et al., 2005), have been associated with reproductive and developmental toxicity in laboratory animals (Butenhoff et al., 2004a; Lau et al., 2004, 2006), and human exposure has been demonstrated (Emmett et al., 2006; Olsen et al., 2004a,b, 2005).
PFOA toxicity studies have been conducted in monkeys, rabbits, mice, and rats (Butenhoff, et al., 2004b; Kennedy et al., 2004; Kudo and Kawashima, 2003; Lau et al., 2004) and epidemiological investigations have been conducted in humans (Emmett et al., 2006; Olsen et al., 2000, 2004a,b, 2005). Although these studies show that pharmacokinetics of the fluorinated compounds vary between species, PFOA has been shown to bioaccumulate in higher trophic organisms including humans (Ehresman et al., 2006; Smithwick et al., 2006; Tomy et al., 2004). Although PFOA's biomagnification potential is less than that of the structurally related perfluorooctane sulfonate (PFOS), bioaccumulation was shown to occur in an aquatic marine food web (Tomy et al., 2004). Furthermore, numerous perfluorinated chemicals (PFCs) such as PFOS (Lau et al., 2003, 2004; Luebker et al., 2005a,b; Thibodeaux et al., 2003), PFCAs including PFOA (Butenhoff et al., 2004a), and perfluorodecanoic acid (PFDA: Harris and Birnbaum, 1989; Olson and Andersen, 1983) adversely affect both pre- and postnatal development as well as cause neurological and endocrine deficits in laboratory animals (Johansson et al., 2006).
PFOS is developmentally toxic in rats (Butenhoff et al., 2004a; Grasty et al., 2005; Thibodeaux et al., 2003) and PFOA is developmentally toxic in mice (Lau et al., 2004, 2006) resulting in embryonic and postnatal death and growth retardation when animals are administered at least 10 mg PFOS or PFOA/kg/day over total gestation. Interestingly, it is suggested that female rats are unlike humans in their ability to rapidly excrete PFOA (Kudo et al., 2001, 2002). It is hypothesized that rats possess an organic acid transport (OAT)–mediated excretion process that rapidly and efficiently decreases the body burden of PFOA (Kudo et al., 2002) as well as other PFCs. These processes are not maximally expressed until sexual maturity in rats (Buist et al., 2002; Hinderliter et al., 2006). Although the OAT transporters are highly conserved across species it has been shown that modulation and transcription of various isoforms (namely OAT2) are age and sex dependent in rats but less so in mice (Buist and Klaassen, 2004; Buist et al., 2002). In comparison, in the human kidney, OAT2 was the most abundant transcript detected (Bahn et al., 2004); however, gender-based differences in humans have not been definitively elucidated. Based on available data, male (Buist et al., 2002; Hinderliter et al., 2006; Kudo et al., 2002) and female (Lau et al., 2003) rats do not appear to be an appropriate animals model for understanding the human health risk of PFOA exposure (Calafat et al., 2006; Harada et al., 2005).
More recently, the effects of PFOA exposure during pregnancy were investigated in the mouse. PFOA produced dose-dependent effects on the number of resorptions, maternal and fetal weight gain, postnatal survival, and growth deficits at doses between 10 and 30 mg PFOA/kg/day with full litter loss occurring at 40 mg PFOA/kg/day (Lau et al., 2006). Early gestational exposure has been associated with growth reduction, low body weight (BW), and poor postnatal survival of pups (Lau et al., 2006). Lau et al. (2006) concluded that in utero exposure to PFOA provides the major contribution of the associated adverse effects and lactational exposure may be a minor contributor to developmental outcomes. Hinderliter et al. (2005) observed that following gestational exposure, PFOA milk concentrations were proportional to maternal administered dose and thus neonates received a constant dose from the dam until weaning as a result of lactational transport. Secondly, upon repeated dosing with PFOA, approximately 8% of the total administered maternal dose in rats is transferred to the developing neonate via lactation (Hinderliter et al., 2005). Furthermore, neonatal PFOA exposure results in developmental neurotoxic effects. Both PFOA and PFDA adversely affect the cholinergic system in adult mice after neonatal exposure as evidenced by deficits in locomotion and total activity in adult mice, similar to other persistent organic pollutants (Johansson et al., 2006).
Information on the metabolism of 8-2 FTOH in mice is limited and there are no published studies elucidating the maternal-fetal and neonatal distribution of 8-2 FTOH and its oxidation metabolites, PFOA and PFNA. In rats, 8-2 FTOH increases fetal skeletal variations at 500 mg/kg/day with a no observed adverse effect level defined as 200 mg/kg/day (Mylchreest et al., 2005) and behaves like xenoestrogens in vitro (Maras et al., 2006). In order to elucidate plausible exposure scenarios, the objective of this study was to investigate the maternal-fetal transfer of 8-2 FTOH and its metabolites, PFOA and PFNA, as well as the role of lactational transfer and subsequent metabolism of these metabolites in mouse neonates exposed in utero or postnatally (by lactation).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Timed-pregnant CD-1 mice from Charles River Laboratories (Raleigh, NC) were received on gestational day (GD) 5 and allowed to acclimate for 3 days prior to treatment. Pregnant mice were housed in microisolator cages and the rooms maintained between 21°C–26°C and 40–70% relative humidity with a 12-h light/dark cycle. Animals were fed Lab Diet Certified Rodent Chow with both food and water provided ad libitum. All animal studies were conducted in accordance with animal welfare regulations and were approved by the University of Gerogia's Institutional Animal Care and Use Committee (IACUC) committee. Mice received a gavage dose of 30 mg 8-2 FTOH/kg BW in a propylene glycol/water (1:1) vehicle or vehicle control on GD8. The dosing solution was repeatedly homogenized with a hand-held Tissue-Tearor (Biospec Products, Inc., Bartlesville, OK) throughout the dosing period.
Gestational experimental design.
For the in utero exposure study, mice (n = 41) were divided into two groups, control (n = 15) and treated (n = 26). Animals were serially sacrificed at 1 (GD9), 2 (GD10), 5 (GD13), 7 (GD15), and 10 (GD18) days posttreatment. Dams were sacrificed by CO2 asphyxiation, maternal serum, and liver samples were collected and the fetuses were removed from the uterus.
Cross-fostering experimental design.
In the cross-foster study, timed-pregnant mice were divided into control (n = 34) and treated groups (n = 36) and similarly treated with either vehicle or 30 mg/kg BW 8-2 FTOH in vehicle on GD8. Following parturition (postnatal day [PND] 0), half of the treated litters were cross-fostered with dams treated with vehicle control resulting in four treatment groups: no exposure (C/C), in utero exposure only (T/C), postnatal exposure only (via lactation) (C/T), and both in utero and postnatal (lactational) exposure (T/T). These animals were sacrificed on PND 1, 3, and 15, and samples were collected for PFC analysis. The schematic in Figure 1 illustrates the design of both the in utero and postnatal cross-foster study.
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Sera and tissue collection.
Blood was collected via cardiac puncture and placed in 1.2-ml centrifuge tubes on ice prior to centrifugation at 2500 rpm (608 x g) for 15 min. Serum was transferred to a new tube and was stored at – 80°C prior to fluorochemical quantification. Maternal liver as well as placental and fetal tissues were excised for extraction and PFC analysis by gas chromatography coupled with mass spectrometry (GC/MS). Neonatal serum and liver (n
5 per dam) were collected on PND3 and PND15. All tissues were weighed on a Mettler PM4000 balance and immediately flash frozen with a combination of liquid nitrogen and dry ice and stored at – 80°C until analysis.
Chemicals.
All chemical reagents used in this study were obtained at the highest purity, greater than 98% as determined by the supplier. 8-2 FTOH (CAS Number 678-39-7) was purchased from Oakwood Research Chemicals (West Columbia, SC) as were all other PFCs and their methyl ester counter parts used for comparative analysis. The PFCs used in this study, along with their CAS numbers and structural formulas, are shown in Table 1. Tetrabutylammonium hydrogen sulfate and sodium carbonate were purchased from Aldrich Chemical (Milwaukee WI), as were Diazald, carbitol, and potassium hydroxide.
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PFC analysis.
Both alcohol and nonvolatile acid analyses were conducted via GC/MS using a previously published method (Henderson et al., 2006
). Briefly, samples (250 µl serum, 1.0 g liver or 0.5–1.0 g fetus/neonate) were homogenized in equal volumes of phosphate-buffered saline and extracted following the method of Hansen et al. (2001). After extraction, carboxylic and telomer acids in the extracts were derivatized with diazomethane (Ngan and Toofan, 1991) and analyzed using an HP 6890 Series Gas Chromatograph equipped with a 5973 Mass Selective Detector and an HP 6890 Series Injector (Palo Alto, CA). The mass spectrometer was operated with the electron ionization source in selected ion monitoring mode at m/z values 131, 169, 231, and 331 (Henderson et al., 2006). Furthermore, all data points were confirmed and quantified with liquid chromatography/MS-MS using the method of Powley et al. (2005), incorporating an internal isotopic standard for intralaboratory comparison. All samples were extracted and derivatized on ice and remained capped throughout analysis to prevent the possible evaporative loss of 8-2 FTOH and other volatile intermediates.
Statistical analysis.
Although the gestational and cross-fostering studies were conducted in independent studies, when appropriate, data were combined for statistical analysis such as maternal and fetal/neonatal weight gain. For comparison between control and treated dams, averages (n 5) were analyzed with a general linear model and further significant differences were analyzed with a Students t-test. All statistical analyses were conducted with Statistical Analysis Software (Cary, NC) and a p-value of 0.05 was used as the level of desired significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After treatment with 8-2 FTOH or vehicle, no obvious signs of toxicity such as lethargy or morbidity were observed in pregnant mice. Both control and treated mice gained approximately 25 g during gestation, and there were no significant differences in weight gain between treatment and control groups throughout the study (Fig. 2). Similarly, fetal and neonatal BWs were not affected by treatment with 8-2 FTOH (Tables 2 and 3).
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Average litter size throughout the gestational study was not significantly different between control and treated groups (Table 4). Similarly, average litter size was unaffected by treatment in the cross-foster study with averages for control and treated groups at 13 ± 2 neonates. Litters were examined on GD9, 10, 13, 15, and 18 and no significant differences in implantations, resorptions or fetal deaths were observed in the gestational experiment. Fetuses were examined for gross malformations at GD15 and GD18 in which two nonviable fetuses were excised from 8-2 FTOH treated dams (Table 4). However, in the cross-fostering experiment, 31% of treated dams had at least one nonviable neonate and 27% of the nonviable neonates were characterized as having anencephaly or exencephaly (Table 4). It should be noted that one neonate with anencephaly was viable immediately following birth but was euthanized and is included in the nonviable group. Among viable neonates at term, there were no significant postnatal losses up to PND15.
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Although there were no apparent treatment-related effects on maternal BW gain, 8-2 FTOH treatment did result in significant increases in the maternal liver weight (LW) to BW ratio (relative LW; LW:BW) as well as a transient increase in absolute LW at GD18 (Table 2). Maternal LW:BW was significantly elevated in the 8-2 FTOH treated groups at GD13 through GD18 (Table 2) and this effect continued throughout the postnatal period examined (PND1 through PND15) (Table 3). In comparison, significant increases in LW:BW ratio were seen at both PND5 and PND15 in neonates exposed in utero (T/T and T/C). Furthermore, neonatal absolute LWs were significantly elevated in group T/T at the same time-points (Table 3). Due to limitations in liver size and amount of sample needed for PFC analysis, neonatal livers were not excised from PND1 neonates.
When examining the distribution and metabolism in maternal serum, 8-2 FTOH was no longer detectable in maternal serum or liver 24 h after treatment (GD9). PFOA and PFNA were the only metabolites detected in samples analyzed for 8-2 FTOH, 8-2 fluorotelomer acid (8-2 FTCA), 8-2 fluorotelomer 
,ß-unsaturated acid (8-2 FTUCA), PFOA, and PFNA, all commercially available intermediate metabolites (Fig. 3). Furthermore, no parent compound (8-2 FTOH) was detected in fetal or neonatal tissues at 24 h or throughout the study. Placental concentrations of both PFOA and PFNA were at maximal concentrations 24-h posttreatment and decreased throughout gestation. Placentas from GD18 had a PFOA concentration of 49 ± 13 ng/g, approximately 10-fold less than those determined in serum and liver samples (Figs. 3 and 4).
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) and liver (
and 
) concentrations of PFOA (solid) and PFNA (open) from CD-1 mice orally exposed to 30 mg/kg BW on GD8 (n
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In both the gestational and cross-fostering studies, PFOA was the most abundant metabolite in all tissues examined including those from dams, fetuses, and neonates (Figs. 3–5 and Table 5). Following in utero exposure to 8-2 FTOH, fetal concentrations of both PFOA and PFNA increase temporally reaching maximal concentrations at or near parturition (Fig. 5). Neonatal body burdens of PFOA and PFNA decrease temporally following parturition up to PND15 (Table 5). PFOA and PFNA are approximately 1.5 times more abundant in the serum of neonatal mice compared to liver concentrations based on percent BW (Table 5). Furthermore, evidence for the lactational transport of both PFOA and PFNA is illustrated by the presence of PFOA and PFNA in groups exposed to treated dams only during lactation and elevated concentrations in the group exposed to treated dams both in utero and postnatally when compared to T/C groups (Table 5).
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During gestation, maternal serum concentrations of PFOA were highest at GD9 and decreased temporally up to GD18 (Fig. 3). Similarly, maternal PFNA concentrations were at maximal levels 1-day posttreatment (GD9) and decreased threefold prior to parturition (Fig. 3). In maternal liver at GD18, concentrations of PFOA were approximately six times greater than concentrations of PFNA. Both maternal liver and serum concentrations of PFNA were less than that of PFOA; however, the two metabolites decreased similarly throughout gestation. PFOA and PFNA were transferred to the developing fetuses as early as 24-h posttreatment (GD9) with PFOA concentrations increasing from GD9 to GD18 (Fig. 5). In fetuses, PFNA was above method quantification limits only in the GD18 group but the overall tissue distribution was similar to that of PFOA (Fig. 5).
In the cross-fostering study, maternal serum concentrations of PFOA decreased temporally from PND1 up to PND15 in 8-2 FTOH–treated animals. PFNA concentrations, although less, exhibited a similar trend reaching concentrations below method detection limits at or before PND15 (Table 5). Maternal liver concentrations of PFOA were highest in the cross-fostering experiment immediately following parturition having reached maximal levels during gestation. These concentrations temporally decreased to the lowest concentrations detected at PND15 (Fig. 3). No statistical differences in PFOA concentrations in serum or liver were present between control dams (C/C) or those that received in utero–treated pups (T/C). On PND1, neonatal tissue concentrations of PFOA from in utero–treated groups (T/C and T/T) were not statistically different and concentrations had decreased by PND3, most notably in the group lacking postnatal (i.e., lactational) exposure (T/C, Table 5). This trend continued in both neonatal serum and livers from T/T and T/C groups as PFOA and PFNA concentrations temporally decreased from PND3 to PND15 (Table 5). Neonates appear to be exposed through lactation based on detectible amounts of PFOA in both neonatal serum and liver on PND3 and PND15 in the group (C/T) exposed during the neonatal period only. Throughout the study neither PFOA nor PFNA or any other metabolites were not detected in the control dams, fetuses, or neonates at any time-point.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A major concern of PFCs is their universal detection in mammalian tissues, not only from humans but also from animals such as polar bears living in remote areas (Olsen et al., 2003
; Smithwick et al., 2006). This suggests that atmospheric transport of volatile precursor compounds, such as 8-2 FTOH, may play a role in the remote distribution of the stable nonvolatile acids, PFOA and PFNA. Limited information is currently available on the metabolism of 8-2 FTOH in mammals. In this study, in utero exposure to 8-2 FTOH in mice results in the formation of PFCAs, and the distribution of these metabolites to both the fetus and neonate affects growth and development, as illustrated by significant increases in relative LW (Table 3), post-partum mortality, and increase in neural tube defects (NTDs) in neonates (Table 4).
In vitro metabolism of 8-2 FTOH has been shown to be rapid with parent compound disappearing (78%) by 4-h posttreatment (Martin et al., 2005) while Kudo et al. (2005) illustrated that intermediate metabolites reach peak concentrations at or near 6-h posttreatment in vivo. In the present study, by 24-h posttreatment, no parent compound or intermediate metabolites were identified in maternal or fetal samples. Only the terminal metabolites of 8-2 FTOH metabolism, PFOA and PFNA, were detected in maternal, fetal, and neonatal tissues throughout the duration of this study (GD9 through PND15) (Figs. 3–5 and Table 5).
PFOA is a developmental toxicant in both rats (Staples, 1985) and mice (Lau et al., 2006) but has not been shown to negatively affect reproductive function in rats (Butenhoff et al., 2004a). Furthermore, following chronic oral exposure during pregnancy in rats, PFOA has been detected in fetuses and in neonates confirming placental transfer (Hinderliter et al., 2005; Lau et al., 2006). Because no 8-2 FTOH was detected in fetal or placental tissues as early as 24-h posttreatment, we suggest that the majority of 8-2 FTOH transformation is dependent on maternal metabolism. Our study shows that the maternal metabolism of 8-2 FTOH results in the fetal accumulation of PFOA (GD8–18). Similarly to Lau et al. (2006), we also saw an increase in neonatal mortality and based on Lau et al.'s findings, it is likely due to increasing PFOA concentrations in the developing fetus. However, in contrast, NTDs were seen following in utero treatment with 8-2 FTOH. This may result from proximal transport of parent or intermediate metabolites (i.e., 8-2 FTAL or 8-2 FTCA) and subsequent exposure to the developing fetus. More research is needed to elucidate the mechanisms of 8-2 FTOH developmental toxicity.
Following 8-2 FTOH administration and subsequent metabolism, PFOA accumulates in the fetal compartment reaching maximal concentrations at or near parturition. In contrast, rats show minimal accumulation in the fetus and the amount of PFOA in the fetal compartment is proportional to maternal dose (Butenhoff et al., 2004a). Furthermore, our cross-foster study shows the presence of PFOA in mice exposed only during the neonatal period and suggests that lactational exposure occurs in mice as occurs in rats (Hinderliter et al., 2005).
Mice are more appropriate for elucidating the pharmacokinetics of both PFOA and precursor compounds than are rats. In female rats, PFOA has a half-life of 2–4 h (Kudo et al., 2001; Ohmori et al., 2003) and therefore actual fetal and neonatal exposure is thought to be minimal following maternal dosing (Lau et al., 2004, 2006). The significance of the lack of PFOA-induced reproductive toxicity in rats was questioned by Lau et al. (2006) because chronic dosing results in episodic increases in maternal serum and does not appear to reach steady state (Lau et al., 2006). Based on the calculated half-lives in pregnant dams (14.9 days, from the current study), which are similar to published values for male and female mice (Kudo and Kawashima, 2003), our data support that adult mice are absent sex-mediated excretion processes.
Accumulation of PFOA in the developing mouse fetus can result in fetal body burdens that affect postnatal growth and development. We found that following in utero exposure, 11 of 36 dams had at least one abnormal pregnancy outcome including still births and/or NTDs. Similarly, Lau et al. (2006) determined that the survival of mouse neonates was highly influenced by in utero chronic exposure to PFOA. Since fetal concentrations of PFOA increase temporally after a single oral dose of 8-2 FTOH given to dams, decreased postnatal survival following chronic exposure to PFOA (Lau et al., 2006) may be attributed to the observed increasing concentration during gestation. To our knowledge, this is the first attempt at measuring the maternal-fetal transfer of PFOA following an oral dose of 8-2 FTOH or PFOA.
One potential explanation for the accumulation of PFOA in the fetus after a single exposure is the pH gradient established between the maternal and fetal compartment. Although most cell membranes are generally impermeable to weak, ionized acids (Milne et al., 1958), during pregnancy a pH gradient is established between the maternal and fetal plasma compartments (Nau and Scott, 1986). The more acidic embryonic compartment is able to trap ionized metabolites and therefore may explain the accumulation of PFOA and PFNA noted in this study. Fetal ion-trapping has been demonstrated with 2-methoxyacetic acid (acid dissociation constant [pKa] = 3.57) and 5,5'-dimethylozaxolidine-2,4-dione (pKa = 6.13) both weak acids (O'Flaherty et al., 1992; Terry et al., 1995, respectively). Furthermore, protein binding and possible protein-mediated transport may account for elevated concentrations of these terminal acids in fetal compartments. In a recent attempt to explain varied species half-lives, Andersen et al. (2006) demonstrate the feasibility of a reverse renal proximal tubular transport process being responsible for PFOA retention.
In male mice, 8-2 FTOH metabolism into PFOA causes peroxisome proliferation and affects the normal fatty acid metabolism occurring in the liver via peroxisome proliferation and manifests as increased LW:BW ratio (Kudo et al., 2005). However, it should be noted that increases in relative LWs have also been observed in peroxisome proliferator-activated receptors (PPARs)-alpha nonresponsive species (Butenhoff et al., 2002) and in PPAR-alpha transgenic knockout mice (Yang et al., 2002) suggesting an alternative mechanism of liver enlargement.
In our study, 8-2 FTOH and its subsequent metabolism to PFOA cause an increase in the relative LW (Tables 2 and 3) of both dams and neonates. Our maternal data support the temporal effect on relative LW as the maternal LW:BW ratio was affected as early as GD13, which is 5 days posttreatment, and continued the duration of the study. Secondly, increases in relative LW are induced in the neonates at the earliest time-point investigated suggesting that similar mechanisms of peroxisome proliferation or general liver hypertrophy are occurring following in utero exposure to 8-2 FTOH.
Our study shows that, in mice, 8-2 FTOH is a developmental toxicant and toxicity likely results from its rapid metabolism to PFOA and PFNA and/or exposure to intermediate metabolites of 8-2 FTOH. Maternal exposure to 8-2 FTOH can result in birth defects and neonatal relative LW gain, suggesting exposure to the developing fetus during the critical period of neural tube closure as well as a mechanism involving induced peroxisome proliferation, respectively. The present study supports the mouse as an appropriate animal model for describing the developmental toxicity of 8-2 FTOH and its metabolites as they relate to humans. Based on rat studies, the developmental toxicity of 8-2 FTOH was thought to be negligible (Mylchreest et al., 2005); however, when research animal models such as mice lack active excretion processes, terminal acid metabolites accumulate in the developing fetus following a single oral exposure. Understanding the metabolism 8-2 FTOH and subsequent distribution of PFOA and PFNA as well as the potential developmental toxicity of intermediate metabolites will greatly advance the science behind the human health risk assessment of these fluorotelomer-based chemicals.
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ACKNOWLEDGMENTS |
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The National Exposure Research Laboratory, United States Environmental Protection Agency (USEPA), Athens, GA 30605 (to M.A.S.) and the Interdisciplinary Toxicology Program (ITP), College of Public Health, University of Georgia, Athens, GA 30605 (to W.M.H.) are thanked for their support and funding of this research. Special thanks to Eva McLanahan (ITP, Athens, GA 30605) and Dr J. MacArthur Long (USEPA, Athens, GA 30605) for their endless support and constant motivation.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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